Despite the successes of drug therapy in the US and other industrialized countries, HIV-1/AIDS deaths have continued to rise meteorically in the developing nations of Sub-Saharan Africa, India, and the Far East. The global challenge is now to develop an HIV-1 vaccine that will substantially delay the onset of AIDS, reduce transmission, and prolong life, even if it does not provide complete protection. In order to achieve that goal, effective neutralizing responses must be raised against HIV-1 envelope proteins in the patients. However, the questions of what specific immunogen to use and in what form has been the focus of much debate, due to the ineffectiveness of subunit and peptide vaccines in raising antibodies that react with the viral spikes in their native oligomeric, enveloped forms. Structural biology can play a key role in the characterization, selection and identification of appropriate immunogens. In this proposal, x-ray structural studies of the envelope glycoproteins gpl20 and gp4l, that are responsible for receptor binding, fusion, and cell entry, will define key epitopes that are susceptible to neutralization by human antibodies and small molecule fusion inhibitors. The human antibodies include two (b12, 2G12) of the three most potent and broadly neutralizing antibodies known against primary HIV-1 isolates. The main goal is to utilize these antibodies derived from infected individuals, other V3 loop (19b, 447-52D), and gp4l (NC-i, 4E10) antibodies, and small molecule inhibitors to elucidate the structural details of the regions of the envelope that are critical for biological function, such as the gpl20 V3 loop, CD4 binding site, and the gp41 fusion ectodomain. The specific aims are to crystallize Fab fragments of these antibodies and fusion inhibitors with V3 loop and gp4l peptides, gpl2O, and a disulfide-linked gp140. The structural information will define the extent and nature of potent neutralization sites, probe the structural role of V3 in viral fusion and explain how V3 mutations affect cell tropism. In addition, these structural studies will aid in the design of V3 loop mimetics to inhibit chemokine receptor binding and viral fusion, of peptidomimetics as potential HIV- 1 vaccines, of small molecules to inhibit fusion, and will suggest strategies for how to elicit the elusive types of HIV-1 antibody responses that are required for effective immunity.